Inotropic Antibodies And Therapeutic Uses Thereof

ABSTRACT

Antibodies binding to sites on the alpha-subunit of the (Na++K+)-ATPase increase cardiac contraction of both ventricular myocytes and mouse heart. In particular, antibodies binding to the RSATEEEPPNDD (SEQ ID NO: 1) or DVEDSYGQQWTYEQR (SEQ ID NO: 2) peptides (or isoforms/derivatives thereof) of the alpha-subunit of the (Na++K+)-ATPase, have been found to be highly inotropic. Both the antibodies and the peptides are important for the treatment of human heart failure and other contractile disorders.

This is a divisional application of prior application Ser. No.10/607,583 Filed on Jun. 25, 2003, Ser. No. 12/750,617 filed on Mar. 30,2010, Ser. No. 13/765,367 filed on Feb. 12, 2013, Ser. No. 14/574,310 onDec. 17, 2014, and Ser. No. 15/062,329 on Mar. 7, 2016, which claims thebenefit of U.S. provisional application No. 60/391,514 filed Jun. 25,2002 and U.S. provisional application No. 60/456,879 filed Mar. 21,2003, both of which are hereby incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

Structural regions of the (Na⁺+K⁺)-ATPase that are directly involved inthe regulation of cardiac contractility are provided. Site-specificbinding of these antibodies against the known sequences of the(Na⁺+K⁺)-ATPase significantly increased cardiac contraction of bothisolated rat cardiac myocytes and mouse hearts. These important findingsprovide functional connection between the structure of the(Na⁺+K⁺)-ATPase and cardiac positive inotropy, wherein a novel mode for(Na⁺+K⁺)-ATPase to regulate cardiac function is shown.

BACKGROUND

(Na⁺+K⁺)-ATPase is a target receptor for digitalis and related drugs.Digitalis, digoxin, ouabain and related substances are cardiacglycosides derived from plants. The main pharmacodynamic property ofcardiac glycosides is the ability to increase the force of myocardialcontraction in a dose-dependent manner (positive inotropic effect). Themost probable explanation for the direct positive inotropic effect isthe ability of cardiac glycosides to inhibit membrane-bound(Na⁺+K⁺)-activated adenosine triphosphatase [(Na⁺+K⁺)-ATPase] (Hoffman,B. F. and J. T. Bigger, Jr., “Digitalis and Allied Cardiac Glycosides”in The Pharmacological Basis of Therapeutics, eds. Goodman and Gilman,p. 732, (1980)). The hydrolysis of adenosine triphosphate (ATP) by thisenzyme provides the energy for the sodium potassium pump.

The precise structural region of (Na⁺+K⁺)-ATPase that regulates cardiacfunction is not well understood. Hence, relatively little is known aboutthe endogenous regulation of (Na⁺+K⁺)-ATPase. Catecholamines (Phillis,J. W., Cell, Tissue and Organ Cultures in Neurobiology, pp. 93-97(1978); Horwitz, B. A., Fed Proc., 38:2170-2176 (1979)), thyroid hormone(Smith, T. J. and I. S. Edelman, Fed. Proc., 38:2150-2153 (1979)),aldosterone (Rossier, B. C., et al., Science, 12:483-487 (1987)),linoleic and linolenic acids (Bidard, J. N., et al., Biochem. Biophys.Acta., 769:245 (1984); Tamura, M., et al., J. Biol. Chem., 260:9672(1985); and vanadium (Cantley, L. C., Jr., et al., J. Biol. Chem.,243:7361-7368 (1978)) have all been linked to either direct or indirecteffects on enzyme activity.

Because of their positive inotropic effect, cardiac glycosides (e.g.,digitalis and ouabain) are unrivaled in value for the treatment of heartfailure. Cardiac glycosides are most frequently used therapeutically toincrease the adequacy of the circulation in patients with congestiveheart failure and to slow the ventricular rate in the presence of atrialfibrillation and flutter.

However, cardiac glycosides have narrow therapeutic indices and theiruse is frequently accompanied by toxic effects that can be severe orlethal. The most important toxic effects, in terms of risk to thepatient, are those that involve the heart (e.g., abnormalities ofcardiac rhythm and disturbances of atrio-ventricular conduction).Gastrointestinal disorders, neurological effects, anorexia, blurredvision, nausea and vomiting are other common cardiac glycoside-inducedreactions. Consequently, there is a need in the art for positiveinotropic agents which overcome the disadvantages associated with knownagents, as well as a need for further information on the mechanisms andreceptors associated with cardiac muscle contractility.

It would be highly beneficial to provide patients with a therapeuticcomposition wherein the cardiac regulatory functions of (Na⁺+K⁺)-ATPaseare specifically regulated. Moreover, the identification of the keystructural regions and amino acids of the (Na⁺+K⁺)-ATPase would be ofgreat importance in developing more specific therapeutic molecules,which specifically regulate the cardiac function and differ incharacteristics from currently available digitalis glycosides.

SUMMARY OF THE INVENTION

The present invention provides for the identification of the keyfunctional sites of (Na⁺+K⁺)-ATPase and also of inotropic agents whichdirectly participate in the regulation of cardiac contraction. Inparticular, the inotropic agents are antibodies which bind to thesefunctional sites (epitopes) in the alpha-subunit of (Na⁺+K⁺)-ATPase andinduce a positive inotropic effect.

In particular, the invention provides for isolated and/or purifiedantibodies (including both exogenous and endogenous) which bind to thestructural binding site of (Na⁺+K⁺)-ATPase and regulate (Na⁺+K⁺)-ATPasefunctions. This is of importance in the treatment of heart disease andother muscle contraction diseases. The antibodies of the invention arepolyclonal and/or antisera, monoclonal, and/or humanized antibodies.

The invention also provides for isolated and/or purified antibodies(including both exogenous and endogenous) which specifically recognizethe amino acid sequences comprising RSATEEEPPNDD (SEQ ID NO:1) and/orDVEDSYGQQWTYEQR (SEQ ID NO:1) of the alpha-subunit of (Na⁺+K⁺)-ATPaseenzyme. The binding of the antibodies to these amino acid sequences ofthe alpha-subunit of (Na⁺+K⁺)-ATPase increase cardiac contraction andmyocyte intracellular diastolic and systolic calcium

Preferred antibodies of the invention exert a positive inotropic effectin cardiac myocytes, when they bind to their specific epitopes in thealpha-subunit of (Na⁺+K⁺)-ATPase. The antibodies can be from antisera,polyclonal, monoclonal, and/or humanized antibodies. In a preferredembodiment the antibodies of the invention are used as a therapeuticagent to treat patients suffering from or susceptible to heart diseaseand/or other muscle contraction disorders.

In particular, the invention provides for purified peptides which areused to generate inotropic antibodies when administered in vivo to apatient, suffering from or susceptible to heart disease and/or musclecontractile disorders. These peptides can be administered individuallyor in combination in a pharmaceutically acceptable carrier to a patient.

The invention also provides for isolated and/or purified peptidescomprising the amino acid sequence RSATEEEPPNDD (SEQ ID NO; 1) andDVEDSYGQQWTYEQR (SEQ ID NO; 2) or derivatives thereof (includingisoforms), which are used to generate inotropic antibodies whenadministered in vivo to a patient suffering from or susceptible to heartdisease and/or myocyte contractile disorders. These peptides can beadministered individually or in combination in a pharmaceuticallyacceptable carrier to a patient. Preferred isoforms of such peptides(i.e. comprising the sequence RSATEEEPPNDD (SEQ ID NO; 1) orDVEDSYGQQWTYEQR) (SEQ ID NO; 2) will also will generate such antibodiesand preferably will comprise an amino acid sequence that has only 1, 2,3, 4, 5, 6, 7 or 8 total amino acid differences from the sequence ofRSATEEEPPNDD (SEQ ID NO; 1) or DVEDSYGQQWTYEQR, (SEQ ID NO; 2) morepreferably will comprise an amino acid sequence that has only 1, 2, 3,or 4 total amino acid differences from the sequence of RSATEEEPPNDD (SEQID NO; 1) or DVEDSYGQQWTYEQR (SEQ ID NO; 2).

In accordance with the invention the peptides can be administered inconcentrations in a ratio of 1:1 or in varying ratios to each other asdefined by their concentration.

In another preferred embodiment, the invention provides for vectorswhich encode amino acid sequences which are used to generate inotropicantibodies when administered in vivo to a patient suffering from orsusceptible to heart disease and/or myocyte contractile disorders.Preferably these vectors are under the control of tissue specificpromoters, in particular, cardiac tissue specific. These vectors arealso preferably used in generating sera comprising inotropic antibodiesusing standard methods such as immunizing mammals.

In another preferred embodiment, the invention provides for vectorswhich encode the amino acid sequences RSATEEEPPNDD (SEQ ID NO: 1),DVEDSYGQQWTYEQR (SEQ ID NO: 2) or isoforms (derivatives) thereof, areused to generate inotropic antibodies when administered in vivo to apatient suffering from or susceptible to heart disease and/or myocytecontractile disorders. Preferably these vectors are under the control oftissue specific promoters, in particular, cardiac tissue specific. Thesepeptides are administered as a vaccine to a patient in need of suchtherapy, in order to generate endogenous inotropic antibodies.

The amino acids which are encoded by the vector stimulate the immunesystem to generate antibodies which bind to their epitopes in thealpha-subunit of (Na⁺+K⁺)-ATPase, resulting in increased myocyteintracellular diastolic and systolic calcium. These antibodies, exert apositive inotropic effect in cardiac.

In a preferred embodiment, the invention provides for the therapeuticuse of antisera, polyclonal and monoclonal antibodies and/or humanizedantibodies that specifically bind to amino acid sequences of(Na⁺+K⁺)-ATPase enzyme and modulate the activity of the enzyme, fortreating patients suffering from or susceptible to heart disease and/ormuscle contractile disorders. These antibodies are also used to blockother molecules from binding to drug-interaction sites so that a patientsuffering from heart disorders such as, for example, arhythmia,tachyrhithmia and the like, are useful in regulating cardiaccontraction. The antibodies in this case would also function toeliminate of certain precipitating drugs, including negative inotropicagents (e.g., certain calcium channel blockers and antiarrhythmic drugslike disopyramide), cardiotoxins (e.g., amphetamines) and plasma volumeexpanders (e.g., nonsteroidal antiinflammatory agents andglucocorticoids).

In another preferred embodiment, the invention provides for a method ofgenerating antibodies, wherein binding of the antibodies to an epitopeof the alpha-subunit of (Na⁺+K⁺)-ATPase exerts a positive inotropiceffect in cardiac myocytes, comprising:

generating amino acid sequences corresponding to overlapping peptidefragments, and variants thereof, of the alpha-subunit of (Na⁺+K⁺)-ATPase(including the alpha-subunit of one or more isoforms of(Na⁺+K⁺)-ATPase); and,

obtaining antibodies specific for each peptide fragment by standardmethods; and,

determining the effects of the antibodies on intracellular diastolic andsystolic calcium levels and cell shortenings as compared to controls.

The antibodies produced by this method, when they bind to theirantigenic sites in the alpha-subunit of (Na⁺+K⁺)-ATPase (including thealpha-subunit of one or more isoforms of (Na⁺+K⁺)-ATPase) exert apositive inotropic effect in cardiac myocytes. The antibodies can befrom antisera, polyclonal antibodies, monoclonal antibodies, and/orhumanized antibodies. These antibodies are also used in immunoassays(e.g. RIA, ELISA, etc.) for diagnosing different heart and contractiledisorders.

In another preferred embodiment, the invention provides for a method fordiagnosing heart failure and/or contractile disorders comprising:

isolating heart tissue using standard methods; and,

obtaining cell cultures from the heart tissue using standard methods;and,

allowing the binding of inotropic antibodies to specific epitopes; and,

measuring intracellular diastolic and systolic calcium and cellshortenings.

Preferably, the molecules of the invention are administered to a patientin an effective therapeutic amount to treat the patient suffering fromor susceptible to heart disease and/or muscle contractile disorders.

In another preferred embodiment, the antibodies are administered to apatient (e.g. as a vaccine-type agent or inotrpoic reagent) in atherapeutically effective amount to block other molecules from bindingto drug-interaction sites of (Na⁺+K⁺)-ATPase, wherein the patient issuffering from or susceptible to arhythmias, tachyrhithmias and thelike.

The invention also provides for identifying molecules which target andblock the RSATEEEPPNDD (SEQ ID NO:1) and/or DVEDSYGQQWTYEQR (SEQ IDNO:2) (or isoforms/derivatives thereof) site of alpha-subunit of the(Na⁺+K⁺)-ATPase (including the alpha-subunit of one or more isoforms of(Na⁺+K⁺)-ATPase), comprising:

contacting a myocyte with a desired molecule; and,

measuring the intracellular diastolic and systolic Ca²⁺; and,

measuring cell shortening and heart function; whereby,

identifying molecules useful for therapy of patients suffering from orsusceptible to heart disease and other contractile disorders.

Such molecules are used to generate inotropic antibodies and/or generatepeptide-based vaccines as therapeutic agents in patients suffering fromand/or susceptible to heart disease and other contractile disorders.

The Jianye-2 peptide (comprising or consisting of sequence RSATEEEPPNDD)(SEQ ID NO:1)) as disclosed herein is a particularly preferred peptide.Isoforms (e.g. differing in sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acids, preferably 1, 2, 3, 4, 5, 6, 7 or 8 amino acid differences,more preferably 1, 2, 3, or 4 amino acid differences) of the Jianye-2peptide also are preferred and those amino acid differences may reflectdifferences among species.

The KX-1 peptide (comprising or consisting of sequence DVEDSYGQQWTYEQR(SEQ ID NO:2)) as disclosed herein is a further particularly preferredpeptide. Isoforms (e.g. differing in sequence by 1, 2, 3, 4, 5, 6, 7, 8,9 or 10 amino acids, preferably 1, 2, 3, 4, 5, 6, 7 or 8 amino aciddifferences, more preferably 1, 2, 3, or 4 amino acid differences) ofthe KX-1 peptide also are preferred and those amino acid differences mayreflect differences among species.

Also preferred are antibodies and vaccines made against a region of theH1-H2 domain of the (Na⁺+K⁺)-ATPase (such as the Jianye-2 peptide orisoform thereof).

Additionally preferred are antibodies and vaccines made against a regionof the H7-H8 domain of the (Na⁺+K⁺)-ATPase (such as the KX-1 peptide orisoform thereof).

Other aspects of the invention are described infra.

Definitions

As used herein, “inotropic agents” or “inotropic antibodies” will beused interchangeably and refers to the effect such agents produce, i.e.improves cardiac output by increasing the force of myocardial musclecontraction. “Positive inotropic effect” means that the contractility ofthe cells is enhanced in a dose-dependent manner. A positive inotropiceffect-producing amount of antibodies or peptides of the invention canbe administered to a “mammalian host” (e.g., a human) to treat cardiacmalfunction (e.g., congestive heart failure, paroxysmal atrialtachycardia, atrial fibrillation and flutter). Administration can beeither enteral (i.e., oral) or parenteral (e.g., via intravenous,subcutaneous or intramuscular injection).

As used herein, a “vector” (sometimes referred to as gene delivery orgene transfer “vehicle”) refers to a macromolecule or complex ofmolecules comprising a polynucleotide to be delivered to a host cell,either in vitro or in vivo. The polynucleotide to be delivered maycomprise a coding sequence of interest in gene therapy. Vectors include,for example, viral vectors (such as adenoviruses (“Ad”),adeno-associated viruses (AAV), and retroviruses), liposomes and otherlipid-containing complexes, and other macromolecular complexes capableof mediating delivery of a polynucleotide to a host cell. The inventionalso provides for vectors which are used for treating a patientsuffering from or susceptible heart disease. Vectors can also compriseother components or functionalities that further modulate gene deliveryand/or gene expression, or that otherwise provide beneficial propertiesto the targeted cells. As described and illustrated in more detailbelow, such other components include, for example, components thatinfluence binding or targeting to cells (including components thatmediate cell-type or tissue-specific binding); components that influenceuptake of the vector nucleic acid by the cell; components that influencelocalization of the polynucleotide within the cell after uptake (such asagents mediating nuclear localization); and components that influenceexpression of the polynucleotide. Such components also might includemarkers, such as detectable and/or selectable markers that can be usedto detect or select for cells that have taken up and are expressing thenucleic acid delivered by the vector. Such components can be provided asa natural feature of the vector (such as the use of certain viralvectors which have components or functionalities mediating binding anduptake), or vectors can be modified to provide such functionalities. Alarge variety of such vectors are known in the art and are generallyavailable.

As used herein, the term “administering a molecule to a cell” (e.g., anexpression vector, nucleic acid, peptide, a delivery vehicle, agent, andthe like) refers to transducing, transfecting, microinjecting,electroporating, or shooting, the cell with the molecule. In someaspects, molecules are introduced into a target cell by contacting thetarget cell with a delivery cell (e.g., by cell fusion or by lysing thedelivery cell when it is in proximity to the target cell).

A cell has been “transformed”, “transduced”, or “transfected” byexogenous or heterologous nucleic acids when such nucleic acids havebeen introduced inside the cell. Transforming DNA may or may not beintegrated (covalently linked) with chromosomal DNA making up the genomeof the cell. In prokaryotes, yeast, and mammalian cells for example, thetransforming DNA may be maintained on an episomal element, such as aplasmid. In a eukaryotic cell, a stably transformed cell is one in whichthe transforming DNA has become integrated into a chromosome so that itis inherited by daughter cells through chromosome replication. Thisstability is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the transforming DNA. A “clone” is a population ofcells derived from a single cell or common ancestor by mitosis. A “cellline” is a clone of a primary cell that is capable of stable growth invitro for many generations (e.g., at least about 10).

As used herein, “molecule” is used generically to encompass any vector,antibody, protein, drug and the like which are used in therapy and canbe detected in a patient by the methods of the invention. For example,multiple different types of nucleic acid delivery vectors encodingdifferent types of genes which may act together to promote a therapeuticeffect, or to increase the efficacy or selectivity of gene transferand/or gene expression in a cell. The nucleic acid delivery vector maybe provided as naked nucleic acids or in a delivery vehicle associatedwith one or more molecules for facilitating entry of a nucleic acid intoa cell. Suitable delivery vehicles include, but are not limited to:liposomal formulations, polypeptides; polysaccharides;lipopolysaccharides, viral formulations (e.g., including viruses, viralparticles, artificial viral envelopes and the like), cell deliveryvehicles, and the like.

A “recombinant viral vector” refers to a viral vector comprising one ormore heterologous genes or sequences. Since many viral vectors exhibitsize-constraints associated with packaging, the heterologous genes orsequences are typically introduced by replacing one or more portions ofthe viral genome. Such viruses may become replication-defective,requiring the deleted function(s) to be provided in trans during viralreplication and encapsidation (by using, e.g., a helper virus or apackaging cell line carrying genes necessary for replication and/orencapsidation) (see, e.g., the references and illustrations below).Modified viral vectors in which a polynucleotide to be delivered iscarried on the outside of the viral particle have also been described(see, e.g., Curiel, D T, et al. PNAS 88: 8850-8854, 1991).

Viral “packaging” as used herein refers to a series of intracellularevents that results in the synthesis and assembly of a viral vector.Packaging typically involves the replication of the “pro-viral genome”,or a recombinant pro-vector typically referred to as a “vector plasmid”(which is a recombinant polynucleotide than can be packaged in an manneranalogous to a viral genome, typically as a result of being flanked byappropriate viral “packaging sequences”), followed by encapsidation orother coating of the nucleic acid. Thus, when a suitable vector plasmidis introduced into a packaging cell line under appropriate conditions,it can be replicated and assembled into a viral particle. Viral “rep”and “cap” genes, found in many viral genomes, are genes encodingreplication and encapsidation proteins, respectively. A“replication-defective” or “replication-incompetent” viral vector refersto a viral vector in which one or more functions necessary forreplication and/or packaging are missing or altered, rendering the viralvector incapable of initiating viral replication following uptake by ahost cell. To produce stocks of such replication-defective viralvectors, the virus or pro-viral nucleic acid can be introduced into a“packaging cell line” that has been modified to contain genes encodingthe missing functions which can be supplied in trans). For example, suchpackaging genes can be stably integrated into a replicon of thepackaging cell line or they can be introduced by transfection with a“packaging plasmid” or helper virus carrying genes encoding the missingfunctions.

A “detectable marker gene” is a gene that allows cells carrying the geneto be specifically detected (e.g., distinguished from cells which do notcarry the marker gene). A large variety of such marker genes are knownin the art. Preferred examples thereof include detectable marker geneswhich encode proteins appearing on cellular surfaces, therebyfacilitating simplified and rapid detection and/or cellular sorting. Byway of illustration, the lacZ gene encoding beta-galactosidase can beused as a detectable marker, allowing cells transduced with a vectorcarrying the lacZ gene to be detected by staining, as described below.

A “selectable marker gene” is a gene that allows cells carrying the geneto be specifically selected for or against, in the presence of acorresponding selective agent. By way of illustration, an antibioticresistance gene can be used as a positive selectable marker gene thatallows a host cell to be positively selected for in the presence of thecorresponding antibiotic. Selectable markers can be positive, negativeor bifunctional. Positive selectable markers allow selection for cellscarrying the marker, whereas negative selectable markers allow cellscarrying the marker to be selectively eliminated. A variety of suchmarker genes have been described, including bifunctional (i.e.positive/negative) markers (see, e.g., WO 92/08796, published May 29,1992, and WO 94/28143, published Dec. 8, 1994). Such marker genes canprovide an added measure of control that can be advantageous in genetherapy contexts. “Treatment” or “therapy” as used herein also refers toadministering, to an individual patient, agents that are capable ofeliciting a prophylactic, curative or other beneficial effect in theindividual.

“Gene therapy” as used herein refers to administering, to an individualpatient, vectors comprising a therapeutic gene.

A “therapeutic polynucleotide” or “therapeutic gene” refers to anucleotide sequence that is capable, when transferred to an individual,of eliciting a prophylactic, curative or other beneficial effect in theindividual.

The term “treatment” or grammatical equivalents encompasses theimprovement and/or reversal of the symptoms of heart failure (i.e, theability of the heart to pump blood). “Improvement in the physiologicfunction” of the heart can be assessed using any of the measurementsdescribed herein (e.g., measurement of ejection fraction, fractionalshortening, left ventricular internal dimension, heart rate, etc. inresponse to isoproterenol and/or norepinephrine), as well as any effectupon the patient's survival. A compound which causes an improvement inany parameter associated with heart failure when used in the screeningmethods of the instant invention may thereby be identified as atherapeutic compound.

The term “individual” as used herein refers to vertebrates, particularlymembers of the mammalian species and includes but is not limited to,domestic animals, sports animals, primates and humans; moreparticularly, the term refers to humans.

As used herein, the term “heart failure” is broadly used to mean anycondition that reduces the ability of the heart to pump blood. As aresult, congestion and edema develop in the tissues. Most frequently,heart failure is caused by decreased contractility of the myocardium,resulting from reduced coronary blood flow; however, many other factorsmay result in heart failure, including damage to the heart valves,vitamin deficiency, and primary cardiac muscle disease. Though theprecise physiological mechanisms of heart failure are not entirelyunderstood, heart failure is generally believed to involve disorders inseveral cardiac autonomic properties, including sympathetic,parasympathetic, and baroreceptor responses. The phrase “manifestationsof heart failure” is used broadly to encompass all of the sequelaeassociated with heart failure, such as shortness of breath, pittingedema, an enlarged tender liver, engorged neck veins, pulmonary ralesand the like including laboratory findings associated with heartfailure.

As used herein, “contractile disorders” refers to the abnormalcontractile response of muscle cells as compared to normal muscle cells.Examples of such disorders are arhythmia, tachyrhithmia, and the like.

A “polynucleotide” refers to a polymeric form of nucleotides of anylength, either ribonucleotides or deoxyribonucleotides, or analogsthereof. This term refers to the primary structure of the molecule, andthus includes double- and single-stranded DNA, as well as double- andsingle-stranded RNA. It also includes modified polynucleotides such asmethylated and/or capped polynucleotides.

“Recombinant,” as applied to a polynucleotide, means that thepolynucleotide is the product of various combinations of cloning,restriction and/or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature.

A “gene” refers to a polynucleotide or portion of a polynucleotidecomprising a sequence that encodes a protein. For most situations, it isdesirable for the gene to also comprise a promoter operably linked tothe coding sequence in order to effectively promote transcription.Enhancers, repressors and other regulatory sequences may also beincluded in order to modulate activity of the gene, as is well known inthe art. (See, e.g., the references cited below).

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably to refer to polymers of amino acids of any length. Theseterms also include proteins that are post-translationally modifiedthrough reactions that include glycosylation, acetylation andphosphorylation.

The terms “variant”, “derivative” and “amino acid sequence variant” areused interchangeably and designate polypeptides in which one or moreamino acids are added and/or substituted and/or deleted and/or insertedat the N- or C-terminus or anywhere within the corresponding nativesequence. In various embodiments, a “variant” polypeptide usually has atleast about 75% amino acid sequence identity, or at least about 80%amino acid sequence identity, preferably at least about 85% amino acidsequence identity, even more preferably at least about 90% amino acidsequence identity, and most preferably at least about 95% amino acidsequence identity with the amino acid sequence of the correspondingnative sequence polypeptide.

An “effective amount” is an amount sufficient to effect beneficial ordesired clinical results. An effective amount can be administered in oneor more administrations. The antibodies, peptides or vectors used asvaccines of the present invention can be administered to a patient attherapeutically effective doses to treat (including prevention) heartdisease and/or other muscular contractile disorders. A therapeuticallyeffective dose refers to that amount of the compound sufficient toresult in desired treatment.

As used herein, the term “fragment or segment”, as applied to apolypeptide, will ordinarily be at least about 5 contiguous amino acids,typically at least about 10 contiguous amino acids, more typically atleast about 20 contiguous amino acids, usually at least about 30contiguous amino acids, preferably at least about 40 contiguous aminoacids, more preferably at least about 50 contiguous amino acids, andeven more preferably at least about 60 to 80 or more contiguous aminoacids in length. “Overlapping fragments” as used herein, refer tocontiguous peptide fragments which begin at the amino terminal end of aprotein and end at the carboxy terminal end of the protein. Each peptidefragment has at least about one contiguous amino acid position in commonwith the next peptide fragment, more preferably at least about threecontiguous amino acid positions in common, most preferably at leastabout ten contiguous amino acid positions in common.

As used herein, the term “substantially pure” describes a compound(e.g., a protein or polypeptide) which has been separated fromcomponents which naturally accompany it. Typically, a compound issubstantially pure when at least 10%, more preferably at least 20%, morepreferably at least 50%, more preferably at least 60%, more preferablyat least 75%, more preferably at least 90%, and even more preferably atleast 99%, of the total material (by volume, by wet or dry weight, or bymole percent or mole fraction) in a sample is the compound of interest.Purity can be measured by any appropriate method. In the case ofpolypeptides, for example, purity can be measured by columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Acompound such as a protein is also substantially purified when it isessentially free of naturally associated components or when it isseparated from the native contaminants which accompany it in its naturalstate.

A “heterologous” component refers to a component that is introduced intoor produced within a different entity from that in which it is naturallylocated. For example, a polynucleotide derived from one organism andintroduced by genetic engineering techniques into a different organismis a heterologous polynucleotide which, if expressed, can encode aheterologous polypeptide. Similarly, a promoter or enhancer that isremoved from its native coding sequence and operably linked to adifferent coding sequence is a heterologous promoter or enhancer.

A “substantially pure nucleic acid”, as used herein, refers to a nucleicacid sequence, segment, or fragment which has been purified from thesequences which flank it in a naturally occurring state, e.g., a DNAfragment which has been removed from the sequences which are normallyadjacent to the fragment such as the sequences adjacent to the fragmentin a genome in which it naturally occurs. The term also applies tonucleic acids which have been substantially purified from othercomponents which naturally accompany the nucleic acid, e.g., RNA or DNA,which has been purified from proteins which naturally accompany it inthe cell.

“Homologous”, as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules such as two DNA molecules, or two polypeptide molecules. Whena subunit position in both of the two molecules is occupied by the samemonomeric subunit (e.g., if a position in each of two DNA molecules isoccupied by adenine) then they are homologous at that position. Thehomology between two sequences is a direct function of the number ofmatching or homologous positions. For example, if 5 of 10 positions intwo compound sequences are matched or homologous then the two sequencesare 50% homologous, if 9 of 10 are matched or homologous, the twosequences share 90% homology. By way of example, the DNA sequences 3′ATTGCC 5′ and 3′ TTTCCG 5′ share 50% homology.

A “promoter,” as used herein, refers to a polynucleotide sequence thatcontrols transcription of a gene or coding sequence to which it isoperably linked. A large number of promoters, including constitutive,inducible and repressible promoters, from a variety of differentsources, are well known in the art and are available as or within clonedpolynucleotide sequences (from, e.g., depositories such as the ATCC aswell as other commercial or individual sources).

An “enhancer,” as used herein, refers to a polynucleotide sequence thatenhances transcription of a gene or coding sequence to which it isoperably linked. A large number of enhancers, from a variety ofdifferent sources are well known in the art and available as or withincloned polynucleotide sequences (from, e.g., depositories such as theATCC as well as other commercial or individual sources). A number ofpolynucleotides comprising promoter sequences (such as the commonly-usedCMV promoter) also comprise enhancer sequences. “Operably linked” refersto a juxtaposition, wherein the components so described are in arelationship permitting them to function in their intended manner. Apromoter is operably linked to a coding sequence if the promotercontrols transcription of the coding sequence. Although an operablylinked promoter is generally located upstream of the coding sequence, itis not necessarily contiguous with it. An enhancer is operably linked toa coding sequence if the enhancer increases transcription of the codingsequence. Operably linked enhancers can be located upstream, within ordownstream of coding sequences. A polyadenylation sequence is operablylinked to a coding sequence if it is located at the downstream end ofthe coding sequence such that transcription proceeds through the codingsequence into the polyadenylation sequence.

A “replicon” refers to a polynucleotide comprising an origin ofreplication which allows for replication of the polynucleotide in anappropriate host cell. Examples include replicons of a target cell intowhich a heterologous nucleic acid might be integrated (e.g., nuclear andmitochondrial chromosomes), as well as extrachromosomal replicons (suchas replicating plasmids and episomes).

As used herein, the term “antibody” refers to a polypeptide or group ofpolypeptides which are comprised of at least one binding domain, wherean antibody binding domain is formed from the folding of variabledomains of an antibody molecule to form three-dimensional binding spaceswith an internal surface shape and charge distribution complementary tothe features of an antigenic determinant of an antigen, which allows animmunological reaction with the antigen. Antibodies include recombinantproteins comprising the binding domains, as wells as fragments,including Fab, Fab′, F(ab).sub.2, and F(ab′).sub.2 fragments.

The term “polyclonal” refers to antibodies that are heterogeneouspopulations of antibody molecules derived from the sera of animalsimmunized with an antigen or an antigenic functional derivative thereof.For the production of polyclonal antibodies, various host animals may beimmunized by injection with the antigen. Various adjuvants may be usedto increase the immunological response, depending on the host species.

“Monoclonal antibodies” are substantially homogenous populations ofantibodies to a particular antigen. They may be obtained by anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. Monoclonal antibodies may be obtainedby methods known to those skilled in the art. See, for example, Kohler,et al., Nature 256:495-497, 1975, and U.S. Pat. No. 4,376,110.

As used herein, an “antigenic determinant” is the portion of an antigenmolecule that determines the specificity of the antigen-antibodyreaction. An “epitope” refers to an antigenic determinant of apolypeptide. An epitope can comprise as few as 3 amino acids in aspatial conformation which is unique to the epitope. Generally anepitope consists of at least 6 such amino acids, and more usually atleast 8-10 such amino acids. Methods for determining the amino acidswhich make up an epitope include x-ray crystallography, 2-dimensionalnuclear magnetic resonance, and epitope mapping e.g. the Pepscan methoddescribed by H. Mario Geysen et al. 1984. Proc. Natl. Acad. Sci. U.S.A.81:3998-4002; PCT Publication No. WO 84/03564; and PCT Publication No.WO 84/03506.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, polyclonal antibodiesraised to marker “X” from specific species such as rat, mouse, or humancan be selected to obtain only those polyclonal antibodies that arespecifically immunoreactive with marker “X” and not with other proteins,except for polymorphic variants and alleles of marker “X”. Thisselection may be achieved by subtracting out antibodies that cross-reactwith marker “X” molecules from other species. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select antibodies specifically immunoreactive witha protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988), for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity). Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than 10 to 100 times background.

“Immunoassay” is an assay that uses an antibody to specifically bind anantigen (e.g., a marker). The immunoassay is characterized by the use ofspecific binding properties of a particular antibody to isolate, target,and/or quantify the antigen.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 (which includes FIGS. 1A through 1F) are photographs showing theimmunofluorescent staining of Jianye-2 antibody in rat cardiac myocytesand African green monkey CV-1 cells. FIGS. 1A-1B are photographs of theconfocal image of rat cardiac myocytes in the presence of Jianye-2 (FIG.1A) or with both Jianye-2 antibody and peptide blocker (FIG. 1B). FIG.1C shows the immunofluorescent stainings of Jianye-2 antibody in a groupof CV-1 cells at a magnification of 400.times. FIG. 1D is a photographof a single CV-1 cell image at 3000.times. FIGS. 1E-1F are photographsshowing Jianye-2 antibody staining in the presence of either 1 mMouabain (FIG. 1E) or strophanthidin (FIG. 1F). The results indicate thatJianye-2 antibody binds to its antigenic site of the (Na⁺+K⁺)-ATPase onthe surface of the cell membrane.

FIG. 2 presents electrocardiograms showing results of the time course ofrat heart cell contraction with or without Jianye-2 antibody. Time runsfrom left to right. Column A, shows the baselines of rat heart cellcontraction. Columns B, C, D show the results obtained 10, 20, 30 minafter administration of either buffer (upper panel) or Jianye-2 antibody(lower panel). Enzyme activity was monitored in cell homogenates underthe same experimental condition. The results show that Jianye-2 antibodyenhanced rat heart cell contraction without inhibiting (Na⁺+K⁺)-ATPaseactivity.

FIG. 3 shows that Jianye-2 antibody markedly increased intracellularCa²⁺ contraction and demonstrates that intracellular Ca²⁺ concentrationis involved in the mechanisms of Jianye-2 antibody enhanced heart cellcontraction. The data represent a mean of 12 independent experiments.

FIG. 4 is the dose-dependent contractile response of Jianye-2 antibodyin rat ventricular myocytes. The half maximal contractile response(EC50) is 35 nM. The data represent a mean of four independentexperiments.

FIG. 5 is a schematic representation of pressure-volume loops of theeffect of Jianye-2 antibody on normal mouse heart left ventricle.Jianye-2 antibody (total 150 ml, 6.0 μM in PBS, pH 7.2) was administeredto the mouse heart at a rate of 5 ml/min. The results show that Jianye-2antibody dramatically induced a reversible positive inotropic effect asdemonstrated by the changes in ventricular pressure-volume loops withtime during the cardiac cycle. The results represent one of the sevensimilar experiments.

FIG. 6 shows the time- and concentration-dependent Jianye-2 antibodyeffects on mouse heart contraction in vivo mouse model. At differentfixed concentrations of Jianye-2 antibody as indicated in the figure,the data are plotted in the form of percent of mouse heart contractionas a function of time. Compared with the background control (at 0 min as100%), Jianye-2 antibody increased mouse heart contractions areexpressed as percentages (%) for 5, 10, 15, and 20 min respectivelyafter administration of 3.4 μM (open circles) or 6.0 μM (black circles)of Jianye-2 antibody. Mouse heart contraction was 108(±5.3), 113(±7),116(±10), 122(±9.0) as shown in the open circles, and 115(±9.0),127(±21), 137(±16), and 143(±30) in black circles. First order rateconstants were 1.06 and 2.16%/min for 3.4 μM and 6.0 μM Jianye-2antibody, respectively. The data represent the mean of 5 independentdeterminations.

FIG. 7 shows that the KX-1 antibody enhanced the velocity of shorteningof rat ventricular myocyte and increased the force of contraction of theheart cells. Time runs from left to right. Column A represents thebaselines of rat heart cell contraction. Columns B & C: 5 and 16 minutesfollowing administration of KX-1. The final concentration of KX-1 was0.74 μM. The results indicate that the KX-1 antibody is an inotropicagent.

FIG. 8 represents the changes of intracellular calcium transientfollowing the binding of KX-1 to the (Na⁺+K⁺)-ATPase and indicates thatKX-1 induced heart cell contraction is dependent on intracellular Ca²⁺increase.

FIG. 9 is a graph showing the effect of immunization with KX-1 on therat cardiac heart failure function. The peptide DVEDSYGQQWTYEQR (SEQ IDNO: 2) was injected into the animal as a vaccine to reduce the rate ofprogression of heart failure in the heart failure rat model. TiterMaxGold was used as an adjuvant throughout the experiment. The results showthat endogenous KX-1 antibody generation significantly delayed the rateof the progression of heart failure in heart failure rats (blackcircles). In contrast, cardiac function was significantly depressed inthe control heart failure rat without immunization with KX-1 antigen(open circles).

DETAILED DESCRIPTION OF THE INVENTION

Antibody (Jianye-2 antibody), which recognizes the RSATEEEPPNDD (SEQ IDNO: 1) peptide (H1-H2 domain) of the alpha-subunit of the(Na⁺+K⁺)-ATPase, and KX-1 antibody which recognizes the DVEDSYGQQWTYEQR(SEQ ID NO: 2) peptide (H7-H8 domain) of the alpha-subunit of the(Na⁺+K⁺)-ATPase, have been found to increase the contractility ofventricular myocytes which is important in the treatment of musclecontractile disorders.

In a preferred embodiment, the invention provides for antisera,polyclonal and monoclonal antibodies and/or humanized antibodies thatspecifically bind to amino acid sequences of (Na⁺+K⁺)ATPase, resultingin increased intracellular Ca²⁺ transients and contraction in intactmammalian heart cells and in living mouse heart.

In accordance with the invention, it is preferred that the antibodiesspecifically bind to peptides having an amino acid sequence RSATEEEPPNDD(SEQ ID NO: 1) (the antibody is referred to herein as the “Jianye-2”antibody), and DVEDSYGQQWTYEQR (SEQ ID NO: 2) (the antibody referred toherein as the “KX-1” antibody), mutants or derivatives thereof. Thesepeptides can be conjugated into polypeptides either directly or througha linker. However, the invention is not limited to these sequences butapplies to any sequence in which antibodies can bind resulting incardiac positive inotropy. The Jianye-2 and KX-1 antibodies aredescribed in detail in the Examples which follow.

In a preferred embodiment, the invention provides for the therapeuticuse of antisera, polyclonal and monoclonal antibodies and/or humanizedantibodies that specifically bind to amino acid sequences of(Na⁺+K⁺)-ATPase enzyme and modulate the activity of the enzyme, fortreating patients suffering from or susceptible to heart disease and/ormuscle contractile disorders. These antibodies are also used to blockother molecules from binding to drug-interaction sites so that a patientsuffering from heart disorders such as, for example, arhythmia,tachyrhithmia and the like, are useful in regulating cardiaccontraction. The antibodies in this case would also function toeliminate of certain precipitating drugs, including negative inotropicagents (e.g., certain calcium channel blockers and antiarrhythmic drugslike disopyramide), cardiotoxins (e.g., amphetamines) and plasma volumeexpanders (e.g., nonsteroidal antiinflammatory agents andglucocorticoids).

In another preferred embodiment, antibodies that bind to specificsequences of (Na⁺+K⁺)-ATPase and can produce cardiac positive inotropyare administered to patients in need of such therapy.

In another embodiment, the molecules of the invention are used asdiagnostic agents for heart disease or other contractile disorders, bydetecting, in standard assays, such as ELISAs, RIAs and the like,peptides which are indicative of contractile disorders.

In another preferred embodiment, the invention provides forpharmaceutical compositions comprising peptides which are administeredto patients resulting in the generation of antibodies which recognizesuch peptides resulting in the in vivo generation of inotropicantibodies. Particularly preferred peptides include, but are not limitedto peptides with amino acid sequence RSATEEEPPNDD (SEQ ID NO: 1) and/orDVEDSYGQQWTYEQR (SEQ ID NO: 2), mutants and variants thereof.

In another preferred embodiment, the invention provides for a vaccinewhich codes for amino acids which generate inotropic antibodies whenadministered in vivo to a patient in need of such therapy or treatment.

In accordance with the invention, the antibodies of the invention arealso used as diagnostic agents which detect muscle contractiledisorders, especially, for example, in the heart. In one embodiment, anyof the above-described molecules can be labeled, either detectably, aswith a radioisotope, a paramagnetic atom, a fluorescent moiety, anenzyme, etc. in order to facilitate its detection in, for example, insitu or in vivo assays. The molecules may be labeled with reagents suchas biotin, in order to, for example, facilitate their recovery, and/ordetection.

In another preferred embodiment, where the antibodies or their fragmentsare intended for therapeutic purposes, it is desirable to “humanize”them in order to attenuate any immune reaction. Humanized antibodies maybe produced, for example by replacing an immunogenic portion of anantibody with a corresponding, but non-immunogenic portion (i.e.chimeric antibodies) (Robinson, R. R. et al., Intemational PatentPublication PCT/U.S.86/02269; Akira, K. et al., European PatentApplication 184,187; Taniguchi, M., European Patent Application 171,496;Morrison, S. L. et al., European Patent Application 173,494; Neuberger,M. S. et al., PCT Application WO 86/01533; Cabilly, S. et al., EuropeanPatent Application 125,023; Better, M. et al., Science 240:1041-1043(1988); Liu, A. Y. et al. Proc. Natl. Acad. Sci. USA 84:3439-3443(1987); Liu, A. Y. et al., J. Immunol. 139:3521-3526 (1987); Sun, L. K.et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, Y. etal., Canc. Res. 47:999-1005 (1987); Wood, C. R. et al., Nature314:446-449 (1985)); Shaw et al., J. Natl. Cancer Inst. 80:1553-1559(1988); all of which references are incorporated herein by reference).General reviews of “humanized” chimeric antibodies are provided byMorrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi, V. T. et al.,BioTechniques 4:214 (1986); which references are incorporated herein byreference).

The present invention provides humanized antibody molecules specific forpeptides having an amino acid sequence RSATEEEPPNDD (SEQ ID NO: 1),DVEDSYGQQWTYEQR (SEQ ID NO: 2) or derivatives thereof. However, theinvention is not limited to these sequences but applies to any sequencein which antibodies can bind resulting in cardiac positive inotropy. Inaccordance with the present invention, the humanized antibodiescomprised antigen specific regions in which at least parts of the CDRsof the heavy and/or light chain variable regions of a human antibody(the receptor antibody) have been substituted by analogous parts of CDRsof a murine monoclonal antibody and the humanized antibody canspecifically bind to the same antigen as, for example, the Jianye-2antibody. In a preferred embodiment of the subject invention, the CDRregions of the humanized Jianye-2 is derived from rabbits as describedin the examples which follow. Some of the humanized antibodies describedherein contain some alterations of the acceptor antibody, i.e., human,heavy and/or light chain variable domain framework regions that arenecessary for retaining binding specificity of the donor monoclonalantibody. In other words, the framework region of some embodiments thehumanized antibodies described herein does not necessarily consist ofthe precise amino acid sequence of the framework region of a naturaloccurring human antibody variable region, but contains varioussubstitutions that improve the binding properties of a humanizedantibody region that is specific for the same target as the Jianye-2 orKX-1 antibodies. A minimal number of substitutions are made to theframework region in order to avoid large-scale introductions ofnon-human framework residues and to ensure minimal immunogenicity of thehumanized antibody in humans. The donor monoclonal antibodies of thepresent invention Jianye-2 or KX-1 antibodies, which are specific forthe rat alpha-subunit of (Na⁺+K⁺)-ATPase i.e., RSATEEEPPNDD (SEQ IDNO: 1) and DVEDSYGQQWTYEQR (SEQ ID NO: 2) peptides respectively.

The humanized antibodies compositions of the invention or othertherapeutic agents of the invention may be administered to a patient ina variety of ways. Preferably, the pharmaceutical compositions may beadministered parenterally, i.e., subcutaneously, intramuscularly orintravenously. Thus, this invention provides compositions for parenteraladministration which comprise a solution of the human monoclonalantibody or a cocktail thereof dissolved in an acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., water, buffered water, 0.4% saline, 0.3% glycine and thelike. These solutions are sterile and generally free of particulatematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate, etc. Theconcentration of antibody in these formulations can vary widely, e.g.,from less than about 0.5%, usually at or at least about 1% to as much as15 or 20% by weight and will be selected primarily based on fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected.

Actual methods for preparing parenterally administrable compositions andadjustments necessary for administration to subjects will be known orapparent to those skilled in the art and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th Ed., MackPublishing Company, Easton, Pa. (1980), which is incorporated herein byreference.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin Remington'sPharm. Sci., 15th Ed. (Mack Publ. Co., Easton (1975)).

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds exhibiting large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

Data obtained from cell culture assays and animal studies can be used informulating a range of dosage for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage can varywithin this range depending upon-the dosage form employed and the routeof administration utilized. For any compound used in the method of theinvention, the therapeutically effective dose can be estimated initiallyfrom cell culture assays. A dose can be formulated in animal models toachieve a circulating plasma concentration range that includes the IC₅₀(i.e., the concentration of the test compound, which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to accurately determine useful doses in humans.Levels in plasma can be measured, for example, by high performanceliquid chromatography. A typical daily dose for the therapeuticmolecules of the invention (i.e., antibodies, peptides, vectors encodingpeptides) of the present invention might range from about 1 μg/kg toabout 100 mg/kg of patient body weight or more per day, depending on thefactors mentioned above, preferably about 10 μg/kg/day to 10 mg/kg/day.

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates can beformulated for administration by intra venous or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration can be suitably formulated to give controlled release ofthe active compound. For buccal administration the compositions can takethe form of tablets or lozenges formulated in conventional manner.

The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing or dispersing agents. Alternatively, the active ingredientcan be in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use. The compounds can also beformulated in rectal compositions such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice that can contain one or more unit dosage forms containing theactive ingredient. The pack can for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

The treatment can be monitored by various ways, including echography andelectrocardiograms. “Electrocardiogram” refers to a graphic tracing ofvariations in electrical potential caused by the excitation of the heartmuscle which may be detected at the body surface. “Electrocardiogram”may be abbreviated as “ECG” or “EKG”. The signals may be detected bymeans of metal electrodes attached to the extremities and chest wall,and may then be amplified by a sensitive voltmeter such as theelectrocardiograph. The ECG waveforms are generally labeledalphabetically beginning with the P wave, which represents atrialdepolarization. Approximately 0.16 seconds after the onset of the Pwave, the QRS waves generally appear as a result of depolarization ofthe ventricular muscle, which initiates contraction of the ventricles.Finally, the T wave results from repolarization of the ventricles, whichrepresents the onset of ventricular relaxation. The duration of the “T”wave cycle time is that time in a heart cycle when it is most vulnerableto fibrillation, a condition where the cardiac muscle fiber contractsasynchronously. Electrocardiography is further described in Harrison'sPrinciples of Internal Medicine, Thirteenth Ed., McGraw-Hill, Inc.,Chapter 189, pp. 954-966 (1994), the disclosures of which are herebyincorporated herein by reference, in their entirety.

Echocardiography is the preferred method of monitoring treatment usingthe molecules of the invention. “Echocardiography” (Echo) uses soundwaves to form a picture of the heart valves and heart muscle. The Echomachine sends sound waves to a transducer (a sound sensitive instrument)that is placed on the patient's chest. The sound waves are reflected bythe heart walls (muscle) and heart valves, back to the transducer, whichchanges the sound into a picture. There is no special preparation forthis test. Gel is applied on the patient's chest and a transducer isplaced over the heart area. Heart structures are examined by changingthe direction of the transducer. The sound waves cause no discomfort.When the test is completed the gel is wiped off easily. Thus, an Echodetects the changes and provides information about heart chamber size,wall motion, valve movements, and structural changes in and around theheart.

The invention also provides for vectors which are used for treating apatient suffering from or susceptible heart disease. Vectors can alsocomprise other components or functionalities that further modulate genedelivery and/or gene expression, or that otherwise provide beneficialproperties to the targeted cells. As described and illustrated in moredetail below, such other components include, for example, componentsthat influence binding or targeting to cells (including components thatmediate cell-type or tissue-specific binding); components that influenceuptake of the vector nucleic acid by the cell; components that influencelocalization of the polynucleotide within the cell after uptake (such asagents mediating nuclear localization); and components that influenceexpression of the polynucleotide. Such components also might includemarkers, such as detectable and/or selectable markers that can be usedto detect or select for cells that have taken up and are expressing thenucleic acid delivered by the vector. Such components can be provided asa natural feature of the vector (such as the use of certain viralvectors which have components or functionalities mediating binding anduptake), or vectors can be modified to provide such functionalities. Alarge variety of such vectors are known in the art and are generallyavailable.

The practice of the present invention can suitably employ, unlessotherwise indicated, conventional techniques of molecular biology andthe like, which are within the skill of the art. Such techniques areexplained fully in the literature. See e.g., Molecular Cloning: ALaboratory Manual, (J. Sambrook et al., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989); Current Protocols in Molecular Biology(F. Ausubel et al. eds., 1987 and updated); Essential Molecular Biology(T. Brown ed., IRL Press 1991); Gene Expression Technology (Goeddel ed.,Academic Press 1991); Methods for Cloning and Analysis of EukaryoticGenes (A. Bothwell et al. eds., Bartlett Publ. 1990); Gene Transfer andExpression (M. Kriegler, Stockton Press 1990); Recombinant DNAMethodology (R. Wu et al. eds., Academic Press 1989); PCR: A PracticalApproach (M. McPherson et al., IRL Press at Oxford University Press1991); Cell Culture for Biochemists (R. Adams ed., Elsevier SciencePublishers 1990); Gene Transfer Vectors for Mammalian Cells (J. Miller &M. Calos eds., 1987); Mammalian Cell Biotechnology (M. Butler ed.,1991); Animal Cell Culture (J. Pollard et al. eds., Humana Press 1990);Culture of Animal Cells, 2nd Ed. (R. Freshney et al. eds., Alan R. Liss1987); Flow Cytometry and Sorting (M. Melamed et al. eds., Wiley-Liss1990); the series Methods in Enzymology (Academic Press, Inc.);Techniques in Immunocytochemistry, (G. Bullock & P. Petrusz eds.,Academic Press 1982, 1983, 1985, 1989); Handbook of ExperimentalImmunology, (D. Weir & C. Blackwell, eds.); Cellular and MolecularImmunology (A. Abbas et al., W. B. Saunders Co. 1991, 1994); CurrentProtocols in Immunology (J. Coligan et al. eds. 1991); the series AnnualReview of Immunology; the series Advances in Immunology; OligonucleotideSynthesis (M. Gait ed., 1984); and Animal Cell Culture (R. Freshney ed.,IRL Press 1987).

Preferred vectors for use in the present invention include viralvectors, lipid-based vectors and other vectors that are capable ofdelivering DNA to non-dividing cells in vivo. Presently preferred areviral vectors, particularly replication-defective viral vectors(including, for example replication-defective adenovirus vectors andadeno-associated virus (AAV) vectors. For ease of production and use inthe present invention, replication-defective adenovirus vectors arepresently most preferred.

“Gene delivery,” “gene transfer,” and the like as used herein, are termsreferring to the introduction of an exogenous polynucleotide (sometimesreferred to as a “transgenes”) into a host cell, irrespective of themethod used for the introduction. Such methods include a variety ofwell-known techniques such as vector-mediated gene transfer (by, e.g.,viral infection/transfection, or various other protein-based orlipid-based gene delivery complexes) as well as techniques facilitatingthe delivery of “naked” polynucleotides (such as electroporation, “genegun” delivery and various other techniques used for the introduction ofpolynucleotides). The introduced polynucleotide may be stably ortransiently maintained in the host cell. Stable maintenance typicallyrequires that the introduced polynucleotide either contains an origin ofreplication compatible with the host cell or integrates into a repliconof the host cell such as an extrachromosomal replicon (e.g., a plasmid)or a nuclear or mitochondrial chromosome. A number of vectors are knownto be capable of mediating transfer of genes to mammalian cells, as isknown in the art and described herein. Targeted vectors include vectors(such as viruses, non-viral protein-based vectors and lipid-basedvectors) in which delivery results in transgene expression that isrelatively limited to particular host cells or host cell types. By wayof illustration, therapeutic molecules, for example, nucleic acidsequences encoding for the peptides of the invention, to be delivered toa patient can be operably linked to heterologous tissue-specificpromoters thereby restricting expression to cells in that particulartissue.

“In vivo” gene delivery, gene transfer, gene therapy and the like asused herein, are terms referring to the introduction of a vectorcomprising an exogenous polynucleotide directly into the body of anorganism, such as a human or non-human mammal, whereby the exogenouspolynucleotide is introduced to a cell of such organism in vivo.

The presently preferred means of in vivo delivery, is by injection ofthe vector into a blood vessel directly supplying the myocardium,preferably by injection into a coronary artery. Such injection ispreferably achieved by catheter introduced substantially (typically atleast about 1 cm) within the ostium of one or both coronary arteries orone or more saphenous veins or internal mammary artery grafts or otherconduits delivering blood to the myocardium.

By injecting the vector stock, preferably containing no wild-type virus,deeply into the lumen of one or both coronary arteries (or grafts andother vascular conduits), preferably into both the right and leftcoronary arteries (or grafts and other vascular conduits), andpreferably in an amount of about 10⁷-10¹³ viral particles as determinedby optical densitometry (more preferably 10⁹-10¹¹ viral particles), itis possible to locally transfect a desired number of cells, especiallycardiac myocytes, with genes that encode proteins that regulate cardiaccontraction, such as, for example, the peptides discussed infra, therebymaximizing therapeutic efficacy of gene transfer, and minimizingundesirable effects at extracardiac sites and the possibility of aninflammatory response to viral proteins. Vector constructs that arespecifically targeted to the myocardium, such as vectors incorporatingmyocardial-specific binding or uptake components, and/or whichincorporate inotropic molecules, for example, the peptides describedabove, that are under the control of myocardial-specific transcriptionalregulatory sequences (e.g., ventricular myocyte-specific promoters) canbe used in place of or, preferably, in addition to such directedinjection techniques as a means of further restricting expression to themyocardium, especially the ventricular myocytes. For vectors that canelicit an immune response, it is preferable to inject the vectordirectly into a blood vessel supplying the myocardium as describedabove, although the additional techniques for restricting the potentialfor extracardiac expression can also be employed. Additional referencesdescribing cell types found in the blood vessels, and the structure ofthe vasculature which may be useful in the methods of the presentinvention include the following: W. Bloom & D. Fawcett, A Textbook ofHistology, 10th Ed., (W. B. Saunders Co. 1975). Methods of uses of genetransfer for the treatment or prevention of disease, including heartdisease are described, e.g., Methods in Molecular Biology, Vol. 7: GeneTransfer and Expression Protocols, Murray, E. (ed.), Humana Press,Clifton, N.J. (1991); Mazur et al., Molecular and Cellular Pharmacology,21:104-111, 1994; French, Herz 18:222-229, 1993; Williams, AmericanJoumal of Medical Sciences 306:129-136, 1993; and Schneider and French,Circulation 88:1937-1942, 1993.

“Vasculature” or “vascular” are terms referring to the system of vesselscarrying blood (as well as lymph fluids) throughout the mammalian body.

“Blood vessel” refers to any of the vessels of the mammalian vascularsystem, including arteries, arterioles, capillaries, venules, veins,sinuses, and vasa vasorum.

“Artery” refers to a blood vessel through which blood passes away fromthe heart. Coronary arteries supply the tissues of the heart itself,while other arteries supply the remaining organs of the body. Thegeneral structure of an artery consists of a lumen surrounded by amulti-layered arterial wall.

The invention also provides for methods for identifying peptides andantibodies which are positive inotropic agents. To prepare an antibodythat specifically binds to a region of the (Na⁺+K⁺)-ATPase, purifiedpeptides or their nucleic acid sequences representing the differentsubunits of (Na⁺+K⁺)-ATPase can be used. Using purified peptides ortheir nucleic acid sequences representing the different subunits of(Na⁺+K⁺)-ATPase, antibodies that specifically bind to a desired peptidecan be prepared using any suitable methods known in the art. See, e.g.,Coligan, Current Protocols in Immunology (1991); Harlow & Lane,Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies:Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature256:495-497 (1975). Such techniques include, but are not limited to,antibody preparation by selection of antibodies from libraries ofrecombinant antibodies in phage or similar vectors, as well aspreparation of polyclonal and monoclonal antibodies by immunizinganimals (see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward etal., Nature 341:544-546 (1989)); humanized antibodies; production ofantibodies by any of the methods discussed above. After the antibody isprovided, the specificity of the antibody can be detected using any ofsuitable immunological binding assays known in the art (see, e.g., U.S.Pat. Nos. 4,366,241; 4.376,110; 4,517,288; and 4,837,168). Useful assaysinclude, for example, an enzyme immune assay (EIA) such as enzyme-linkedimmunosorbent assay (ELISA), a radioimmune assay (RIA), a Westem blotassay, or a slot blot assay. These methods are also described in, e.g.,Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai,ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed.1991); and Harlow & Lane, supra.

To determine whether these identified antibodies are positive inotropicagents, standard assays such as those described in the Examples whichfollow can be used. For example, measurement of cell contraction assays;confocal Ca²⁺ imaging; (Na⁺+K⁺)-ATPase activity assays and the like.

In another embodiment peptides that induce production of inotropicantibodies in vivo and in vitro are preferred. The peptides can beindividual peptides, peptides co-administered with adjuvants, peptidescoupled to peptides with different amino acid sequences and/or the samepeptides coupled to each other as repeating units. Also, while peptidesmay be directly coupled to each other, in some cases a small linkersequence or a larger heterolinker molecule may be advantageously used tocouple the two peptides. For example, as the spacer, one or a few, up toabout 5, preferably, up to about 3, neutral amino acids, such asglycine, may be used to link the peptides. A preferred spacer peptide isGGG, however, the spacer may be made larger or smaller and altered toinclude other molecules besides the amino acid glycine. As examples ofheterolinkers may be made of, for example,N-succinimidyl-3-(2-pyridylthi-o)propinate (SPDP),m-maleimidobenzoyl-N-hydroxy-succimide (MBS) as well as any of the otherreagents employed to link peptides. When the peptides are not directlybonded the linking group will generally and preferably be any divalentlinking group. The linking group may be cleavable or non-cleavable underphysiological conditions or by appropriate inducement.

Although the total number of amino acids in the conjugated polypeptideis not particularly critical, from a practical aspect, the minimumnumber of amino acids, including any amino acid spacers or linkers, willgenerally be at least about 10 or 12, preferably at least about 20, toobtain adequate antigen presentation and immunogenicity up to about 100amino acids.

The polypeptides of this invention may be used as a vaccine eitherprophylactically or therapeutically. When provided prophylactically thevaccine is provided in advance of any evidence of muscular contractiledisorders. Antibodies are produced against the peptides which areinotropic. The prophylactic administration of the invention vaccineshould serve to prevent or attenuate, for example cardiac musclecontractile disorders. In a preferred embodiment a human, at high riskfor heart muscle contractile disorders is prophylactically treated witha vaccine of this invention. When provided therapeutically, the vaccineis provided to enhance the patient's own antibody response to producethe desired inotropic antibodies.

While it is possible for the immunogenic polypeptide, which may or maynot be conjugated, to be administered in a pure or substantially pureform, it is preferable to present it as a pharmaceutical composition,formulation or preparation.

The formulations of the present invention, both for clinical and forhuman use, comprise a conjugated polypeptide as described above,together with one or more pharmaceutically acceptable carriers and,optionally, other therapeutic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any method well-known in the pharmaceutical art.

In general, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product into the desired formulation.

Formulations suitable for any route of administration may be used, suchas, for example, intravenous, intramuscular, subcutaneous,intraperitoneal, nasal, oral, rectal, vaginal, etc. Generally, theformulations will comprise sterile aqueous solutions of the activeingredient with solutions which are preferably isotonic with the bloodof the recipient. Such formulations may be conveniently prepared bydissolving solid active ingredient in water containing physiologicallycompatible substances such as sodium chloride (e.g. 0.1-2.0M), glycine,and the like, and having a buffered pH compatible with physiologicalconditions to produce an aqueous solution, and rendering the solutionsterile. These may be present in unit or multi-dose containers, forexample, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer.Illustrative stabilizers include polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, and organic acids which maybe used either on their own or as admixtures. These stabilizers, whenused, are preferably incorporated in an amount of about 0.1 to about10,000 parts by weight per part by weight of immunogen. If two or morestabilizers are to be used, their total amount is preferably within therange specified above. These stabilizers are used in aqueous solutionsat the appropriate concentration and pH. The specific osmotic pressureof such aqueous solutions is generally in the range of about 0.1 toabout 3.0 osmoles, preferably in the range of about 0.3 to about 1.2.The pH of the aqueous solution is adjusted to be within the range ofabout 5.0 to about 9.0, preferably within the range of 6-8. Informulating the immunogen of the present invention, anti-adsorptionagent may be used.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the conjugatedpolypeptide. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate theconjugated polypeptide into particles of a polymeric material such aspolyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylenevinylacetate copolymers. Alternatively, instead of incorporating theseagents into polymeric particles, it is possible to entrap thesematerials in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxy-methylcellulose or gelatin-microcapsules andpoly(methylmethacrylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combinedwith typical carriers, such as lactose, sucrose, starch, talc, magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others. Thesecarriers may likewise be used for preparing to be administered via othercavities, e.g., nasal, rectal, etc.

The conjugated polypeptides of the present invention may be supplied inthe form of a kit, alone, or in the form of a pharmaceutical compositionas described above.

As noted above, the administration of the vaccine of the presentinvention may be for either a prophylactic or therapeutic purpose. Whenprovided therapeutically, the immunogen is provided at (or after) theonset of the disease or at the onset of any symptom of the disease. Thetherapeutic administration of the immunogen serves to attenuate thedisease.

The present invention, therefore, provides antigenic conjugatedpolypeptides, which provide powerful vaccines for eliciting immuneresponses for production of inotropic antibodies in vivo.

The conjugated polypeptides, which may be prepared by conventional solidphase peptide synthesis or other conventional means for peptidesynthesis, however, the peptides may also be prepared by geneticengineering techniques. The DNA sequences coding for the peptides ofthis invention can be prepared by any of the well known techniques forrecombinant gene technology. For example, reference can be made to thedisclosure of recombinant proteins and peptides in U.S. Pat. No.5,142,024 and the body of literature mentioned therein, the disclosuresof which are incorporated herein by reference thereto.

The following non-limiting examples are illustrative. All documentsmentioned herein are hereby incorporated by reference.

EXAMPLES

In the following examples, the following materials and methods wereemployed.

Materials and Methods

Materials.

All reagents were purchased from Sigma Chemical Co., unless specified.Highly purified dog kidney (Na⁺+K⁺)-ATPase was a gift from Dr. JackKyte.

Antibody Preparation.

The RSATEEEPPNDD (SEQ ID NO:1) and DVEDSYGQQWTYEQR (SEQ ID NO:2)peptides were synthesized according to the protein sequence reported(Schneider, J. W., Mercer, R. W., Caplan, M., Emanuel, J. R., Sweadner,K. J., Benz, E. J., Levenson, R. (1985) Proc. Natl. Acad. Sci. U.S.A.82, 6357-6361; Xie, Z., Li, H., Liu, G., Wang, Y., Askari, A., Mercer,R. W. (1994) Cloning of the dog Na/K-ATPase alpha 1 subunit. The NaPump. (Bamberg, S., and Schoner, W., Eds), pp. 49-52, Springer-Verlag,New York, N.Y.; Shull, M. M., Lingrel, J. B. (1987) Proc. Natl. Acad.Sci. U.S.A. 84, 4039-4043). The polyclonal Jianye antibody was generatedin New Zealand White rabbits using KLH as a peptide carrier (Genemed).The immunoglobulins (IgG) were purified through an affinity columndirected against the same synthetic peptide of the (Na⁺+K⁺)-ATPase.Purified antibodies recognize both denatured (by Western blots) andnative (Na⁺+K⁺)-ATPase (by immunocytostaining). Synthetic peptides werealso utilized as the specific peptide blockers for the antibodies.

Isolation of Cardiac Myocytes:

Ventricular cardiac myocytes were isolated from adult Sprague-Dawleyrats (2-3 months old; weight 225-300 g) using standard enzymatictechniques. Briefly, following anesthesia (sodium pentobarbital, 100mg/kg), the heart was quickly removed from the chest and aortic perfusedat constant pressure (100 cmH₂O) at 37° C. for 3 minutes with aCa²⁺-free bicarbonate-based buffer containing 120 mM NaCl, 5.4 mM MgSO₄,1.2 mM NaH₂PO₄, 5.6 mM glucose, 20 mM NaHCO₃, and 5 mM taurine, in thepresence of O₂ (95%)-CO₂ (5%). The enzymatic digestion was initiated byadding collagenase (Worthington Type II, 1 mg/ml) to the perfusionsolution. Calcium (50 μM) was added to the enzyme solution when theheart became swollen. About 7 minutes later, the left ventricle wasquickly removed, cut into several pieces, and further digested on ashaker (60-70 rpm) for 10 minutes in the same enzyme solution. Thesupernatant containing the dispersed myocytes was filtered into a testtube and gently centrifuged at 500 rpm for 1 minute. The cell pellet wasthen promptly resuspended in a solution containing 0.125 M Ca²⁺. Thesupernatant was aspirated after the myocytes were pelleted by gravityfor 10 minutes and the myocytes were then resuspended in a solutioncontaining 0.25 mM Ca²⁺. The shake-harvest procedure was repeatedseveral times until all of the pieces were digested. For freshlyisolated cells, myocytes were suspended in HEPES-buffer consisting of 1mM CaCl₂, 0.137 mM NaCl, 5.4 mM KCl, 15 mM dextrose, 1.3 mM MgSO₄, 1.2mM NaH₂PO₄, and 20 mM HEPES, pH 7.4.

Measurement of Mice Cardiac Contraction In Vivo:

Male wild-type mice (CD1, Charles River, 32-40 g) were utilized for thisstudy. Mice In vivo cardiac functions were assessed by pressure-volumecatheter in anesthetized mice. Briefly, mice were induced with 5%isofluorane, anesthetized with an intraperitoneal injection of urethane(300-500 mg/kg), etomidate (5 mg/kg) and morphine (0.5 mg kg⁻¹), andintubated with a blunt 19G needle inserted via tracheostomy. Additionalsmall dose was given when increase in heart rate or blood pressure wasobserved in response to tail pinch with forceps. Ventilation wasinitiated with 100% oxygen using a custom-designed, constant flowventilator delivering a tidal volume of 6.7 μL/kg at 120 breaths permin. The left external jugular vein was cannulated with a 30G needleconnected to an infusion pump. Modest volume expansion was provided (150μL of 12.5% human albumin) at 50 μL/min. Following stabilization, alateral incision was made at the xyphoid cartilage to expose the leftventricular (LV) apex. The 1.4F pressure-volume catheter (SPR-839,Millar Instruments Inc., Houston, Tex., USA) was inserted via an apicalneedle puncture with a 26G needle, and advanced along the cardiac longaxis. A 2F pacing catheter (NuMed, Nicholville, N.Y., USA) was placed inthe esophagus, dorsal to the left atrium. Atrium was paced using 5-7 V,2 ms pulses (SD25, Grass Instruments, Quincy, Mass., USA). Calibrationof the volume signal was performed using a 5-10 μL bolus of 30%hypertonic saline injected into the jugular vein to determine the signaloffset and ultrasound flow probe (AT01 RB, Transonic Systems Inc.)placed around the thoracic aorta to determine signal gain. Data weredigitized at 2 kHz and stored to disk for off line analysis.

Mice cardiac atria pacing were maintained at constant beating (600beats/min). The hearts were infused with PBS (5 μl/min) for 10 min priorfor administration of SSA78 infusion (5 μl/min) for 30 minutes andfollowing by 10 min PBS washout at 5 μl/min. Control experiments showedthat the vehicle (PBS), at the experimental infusion rates, has noeffect on cardiovascular performance. Indices of myocardial systolic anddiastolic performance were derived from pressure-volume data obtainedboth at steady-state (every minute) and during transient loading of theheart with direct occlusion of the inferior vena cava (IVC) (every 5minutes). Steady-state indices were derived from 10 consecutive averagedbeats. Cardiac preload was indexed as the left ventricular end-diastolicvolume (EDV) and end-diastolic pressure (EDP). Cardiac afterload wasevaluated as effective arterial elastance (Ea; ratio of LV systolicpressure to stroke volume). This parameter is not preload dependent andhad been validated to closely approximate total afterload, whichincorporates systemic vascular resistance, aortic impedance, and thereflected wave properties of the vasculature. Myocardial contractilitywas indexed by cardiac output (CO), dPdt max, dPdt max normalized toinstantaneous developed pressure (dPdt max/IP), the load-independentend-systolic pressure-volume relationship (Ees) and preload recruitablestroke work (PRSW). Diastolic performance was measured by dPdt min andthe time constant of ventricular relaxation.

Measurement of Cell Contraction

Cardiac myocytes were isolated from wild-type adult Sprague-Dawley rats,using a standard enzymatic method described previously (Xu, K. Y., etal., (2002) BBRC, 289:167-172). Isolated myocytes were suspended in abuffer containing (in mM) 137 NaCl, 5.4 KCl, 15 dextrose, 1.3 MgSO₄, 1.2NaH₂PO₄, 1 CaCl₂, and 20 HEPES, pH 7.4. To measure cell contractility,cardiac myocytes were placed on an inverted microscope (Zeiss modelIM-35), bathed with a HEPES-buffered solution, and electricallystimulated under 0.5 Hz at room temperature. The designated reagentswere added when the baseline contraction was stabilized after 10-15 minconstant pacing. Cell length was monitored from the bright-field image(650 nm to 750 nm red light illumination) by an optical edge-trackingmethod using a photodiode array (model 1024 SAQ, Reticon) with a 3-mstime resolution. The contraction amplitude was indexed by the percentageshortening of cell length.

Measurement of Intracellular Ca²⁺ Transients

Rat cardiac myocytes were loaded with 50 μg of a cell permeablefluorescent Ca²⁺ probe, Indo-1/acetoxymethyl-ester (Indo-1/AM, MolecularProbes, Inc., Eugene, Oreg.) for 10 min and then resuspended inHEPES-buffered solution in the presence of 1 mM Ca²⁺ and stored in thedark at room temperature for 60 min before be utilized for theexperiments. The Indo-1 loaded cells were placed on the stage of amodified inverted microscope (model IM-35; Carl Zeiss, Inc., Thomwood,N.Y.) equipped for simultaneous recording of Indo-1 flrorescence andcell length. Cells were electrically paced at 0.5 Hz at room temperatureand the excitation wavelength was selected by a 350-nm interferencefilter (bandwidth 10 nm; Oriel Corp., Stratford, Conn.). The ratio ofemission intensity at 410 nm to that at 490 nm was computed offline asan index of intracellular Ca². (Ca_(i) ²⁺) transient. The peak amplitudeof the transient is defined as the difference of the 410:490fluorescence ratio before and after electrical stimulation with orwithout antibody.

Isolation of Sarcolemmal Vesicles and Purification of (Na⁺+K⁺)-ATPase:

Rat cardiac sarcolemmal (SL) vesicles were isolated from rat heartmuscle by sucrose flotation method. The vesicles were tested withsaponin and were predominately right-side-out in orientation.(Na⁺+K⁺)-ATPase was purified as described previously (Kyte, J. (1971)Biochemistry, 246:4157-4165). Briefly, the SL vesicles (4.4 mg/ml) weretitrated with 0.58 mg/ml of SDS in the presence of 2 mM ATP at20.degree. C. for 30 min. The SDS titrated fractions were then loaded onthe top of a sucrose (W/W) step gradient constructed with 10 ml of 37.3%(bottom step), 20 ml of 28.8%, and 10 ml of 15% in a Ti 60 tube, andcentrifuged at 40,000 rpm for 90 min. The fractions that contain(Na⁺+K⁺)-ATPase (between 37.3 and 28.8% on the sucrose gradient) werecarefully collected and sedimented at 40,000 rpm for 60 minutes. Thepurified enzyme was resuspended in a sucrose (250 mM)/histidiniumchloride (30 mM) buffer, pH 7.2, quick-frozen in liquid nitrogen andstored at 70.degree. C. Highly purified dog kidney (Na⁺+K⁺)-ATPase was agift from Dr. Jack Kyte.

Determination of (Na⁺+K⁺)-ATPase Activity:

The enzymatic activity was determined as described previously (Kyte J.,et al., (1987) Biochemistry, 26:8350-8360) with modifications. Briefly,purified rat or dog (Na⁺+K⁺)-ATPase was incubated with or without Jianyeor ouabain in the presence of 100 mM Na.+ for 30 min at roomtemperature. The reaction was initiated by adding 3 mM MgATP and 20 mMK⁺ in a final volume of 0.25 ml at 37° C. for 30 min and terminated byadding 0.75 ml quench solution and 0.025 ml developer. The color wasallowed to develop for 30 min at room temperature and the concentrationof phosphate was then determined at 700 nm using a spectrophotometer.

Echocardiography

Echocardiography (Echo) uses sound waves to form a picture of the heartvalves and heart muscle. The Echo machine sends sound waves to atransducer (a sound sensitive instrument) that is placed on the animal'schest. The sound waves are reflected by the heart walls (muscle) andheart valves and back to the transducer, which changes the sound into apicture. Gel is applied on the rat chest and a transducer is placed overthe heart area. Heart structures are examined by changing the directionof the transducer. The sound waves cause no discomfort. Thus, an Echodetects the changes and provides information about heart chamber size,wall motion, valve movements, and structural changes in and around theheart.

Example 1 Jianye-2 Inotropic Antibody

Jianye-2 antibody, which specifically recognizes the RSATEEEPPNDD (SEQID NO:1) peptide of the alpha-subunit of the (Na⁺+K⁺)-ATPase, has beenfound to increase the contractility of isolated rat ventricularmyocytes. FIG. 1 shows the results obtained with immunofluorescentstaining of Jianye-2 antibody in rat cardiac myocytes and African greenmonkey CV-1 cells. Confocal image of rat cardiac myocytes in thepresence of Jianye-2, shown in FIG. 1A or with both Jianye-2 antibodyand peptide blocker (FIG. 1B). FIG. 1C shows the immunofluorescentstainings of Jianye-2 antibody in a group of CV-1 cells at amagnification of 400.times. FIG. 1D shows the CV-1 cell image at 3000times. Jianye-2 antibody staining in the presence of either 1 mM ouabain(FIG. 1E) or strophanthidin (FIG. 1F). The results reveal that ouabainand strophanthidin compete with the Jianye-2 antibody binding siteindicating that Jianye-2 antibody specifically binds to the(Na⁺+K⁺)-ATPase on the extracellular surface of the cell membrane. FIG.2 shows results of the time courses of rat heart cell contraction withor without Jianye-2 antibody. Time runs from left to right. Column A,shows the baselines of rat heart cell contraction. Columns B, C, D showthe results obtained 10, 20, 30 min after administration of eitherbuffer (upper panel) or Jianye-2 antibody (lower panel, 85 nM). Enzymeactivity was monitored in cell homogenates under the same experimentalcondition. The results show that Jianye-2 antibody enhanced rat heartcell contraction without inhibiting (Na⁺+K⁺⁾-ATPase activity. FIG. 3shows that Jianye-2 antibody markedly increased intracellular Ca²⁺contraction and demonstrates that intracellular Ca²⁺ concentration isinvolved in the mechanisms of Jianye-2 antibody enhanced heart cellcontraction. The data represent a mean of 12 independent experiments.FIG. 4 shows the dose-dependent contractile response of Jianye-2antibody in rat ventricular myocytes. The half maximal contractileresponse (EC50) is 35 nM. The data represent a mean of four independentexperiments.

FIG. 5 is a schematic representation of pressure-volume loops of theeffect of Jianye-2 antibody on normal mouse heart left ventricle.Jianye-2 antibody (total 150 ml, 6.0 μM in PBS, pH 7.2) was administeredto the mouse heart at a rate of 5 ml/min. The results show that Jianye-2antibody dramatically induced a reversible positive inotropic effect asdemonstrated by the changes in ventricular pressure-volume loops withtime during the cardiac cycle. The results represent one of the sevensimilar experiments. FIG. 6 shows the time- and concentration-dependentJianye-2 antibody effects on mouse heart contraction in vivo model. Atdifferent fixed concentrations of Jianye-2 antibody as indicated in thefigure, the data are plotted in the form of percent of mouse heartcontraction as a function of time. Compared with the background control(at 0 min as 100%), Jianye-2 antibody increased mouse heart contractionsare expressed as percentages (%) for 5, 10, 15, and 20 min respectivelyafter administration of 3.4 mM (open circles) or 6.0 mM (black circles)of Jianye-2 antibody. Mouse heart contraction was 108(±5.3), 113(±7),116(±10), 122(+9.0) as shown in the open circles, and 115(+9.0),127(±21), 137(±16), and 143(±30) in black circles. First order rateconstants were 1.06 and 2.16%/min for 3.4 mM and 6.0 mM Jianye-2antibody, respectively. The data represent the mean of 5 independentdeterminations.

Example 2 KX-1 Inotropic Antibody

Antibody (KX-1), which recognizes DVEDSYGQQWTYEQR (SEQ ID NO:2) peptideof alpha-subunit of the (Na⁺+K⁺)-ATPase, has been found to increase thecontractility of isolated rat heart cells. Vaccination of its specificpeptide-antigen in heart diseased animal model significantly decreasedthe progression of heart failure. This KX-1 antibody, its peptidevaccine, and monoclonal and humanized antibodies are useful fortreatment of heart failure and other contractile disorders. FIG. 7 showsthat the KX-1 antibody enhanced the velocity of shortening of ratventricular myocyte and increased the force of contraction of the heartcells. The results indicate that the KX-1 antibody is an inotropicagent.

FIG. 8 represents the changes of intracellular calcium transientfollowing the binding of KX-1 to the (Na⁺+K⁺)-ATPase and indicates thatKX-1 induced heart cell contraction is dependent on intracellular Ca²⁺increase. FIG. 9 reveals that the DVEDSYGQQWTYEQR (SEQ ID NO:2) peptidecan be utilized as a vaccine to generation of endogenous KX-1 antibodyin heart failure rats and the results show that endogenous KX-1 antibodydelayed the rate of progression of heart failure in live heart failurerat animal models. In contrast, cardiac function was significantlydepressed in the control heart failure rat without immunization withKX-1 antigen.

What is claimed is:
 1. A method of producing a positive inotropic effectin a mammalian host comprising administering to said mammalian host atherapeutically effective amount of a synthetic peptide consisting ofthe amino acid sequence of RSATEEEPPNDD (SEQ ID NO:1) or isoformthereof, wherein an endogenous antibody is generated through thesynthetic peptide antigen and the endogenous antibody binds the alphasubunit of the (Na⁺+K⁺)-ATPase to increase intracellular calcium,wherein the endogenous antibody enhances the velocity of shortening ofthe ventricular myocyte and increases the force of contraction of theheart cells, wherein the mammalian host suffers from a heart disease inneed of an enhancement of the velocity of shortening of the ventricularmyocyte and an increase in the force of contraction of the heart cells.2. The method of claim 1, wherein the peptide or isoform is administeredindividually or in combination with one or more pharmaceuticallyacceptable carrier to the host.
 3. The method of claim 1, wherein themethod further comprises monitoring the mammal's cardiac contractionsfor a positive inotropic effect.